POS0098 LINC01871, IMPLICATED IN SJÖGREN’S DISEASE PATHOGENESIS, IS REGULATED BY INTERFERON-G AND CALCINEURIN SIGNALING
Authors: M. L. Joachims, B. Khatri, C. Li, K. L. Tessneer, J. Ice, A. M. Stolarczyk, N. Means, K. Grundahl, S. Glenn, J. Kelly, D. Lewis, L. Radfar, D. Stone, J. Guthridge, J. A. James, R. H. Scofield, G. B. Wiley, J. Wren, P. M. Gaffney, C. Montgomery, K. Sivils, A. Rasmussen, A. D. Farris, I. Adrianto, C. Lessard
Journal: Annals of the Rheumatic Diseases (2022)

Background Sjögren’s disease (SjD) is an autoimmune disease characterized by exocrine gland dysfunction. Long non-coding RNAs (lncRNAs) are a functionally diverse class of non-protein coding RNAs that are longer than 200 nucleotides. Our previous study using whole blood RNA-seq found that lncRNA, LINC01871 , is overexpressed in SjD relative to controls [1]. CRISPR-Cas9 targeting in HSB2 T cells yielded a LINC01871 -/- clone with altered expression of many genes implicated in immune regulation [1]. Objectives The goal of this study was to analyze the gene expression perturbations resulting from the loss of LINC01871 and to characterize the regulation of LINC01871 in both the LINC01871 -/- clone and primary human T cells in response to immune stimuli. Methods Flow cytometry and LegendPlex bead assays were used to compare surface and secreted protein expression changes, respectively, in LINC01871 -/- cells and the parental HSB2 cells. Parental HSB2 T cells, LINC01871 -/- cells, Kasumi-3 myeloid cells, and primary human T cells were stimulated in vitro and changes in gene expression were measured over time using qRT-PCR. Responses to interferons (IFN) were assessed using universal type I IFN (IFNα) or IFNγ. TCR signaling responses were assessed using PMA/Ionomycin (PMA/I) or anti-CD3/CD28 stimulations in the presence or absence of the calcineurin inhibitor, FK506. Results Previous RNA-seq analysis found 1166 differentially expressed (DE) transcripts (log 2 FC ≥1 or ≤-1; p adj ≤0.05) in LINC01871 -/- cells compared to parental HSB2 cells, including many prominent immune regulatory genes. Changes in the basal expression of 7 proteins in LINC01871 -/- cells were confirmed using flow cytometry (significantly decreased: CD8a (p=0.0004), CD30 (p=0.0008), CXCR3 (p=0.037), T-Bet (p=0.0002), and Aiolos (p=0.02); significantly increased: CD226 (p=0.0059) and CD44 (p=0.024)). Analysis of LINC01871 -/- cells revealed a growth inhibition in LINC01871 -/- cells (p=0.0014 at 72h), in which multiple secreted growth and adhesion factors were significantly reduced: GM-CSF (p=2.0e-06), M-CSF (p=2.7e-09), IGBPF4 (p=1.2e-07), s-ICAM1 (p=0.015), MMP9 (p=3.0e-14), and MMP2 (p=6e-08). In contrast, the IL-6 cytokine family member, LIF, was significantly increased in LINC01871 -/- cells (p=1.2e-07). Because HSB2 cells were not responsive to all IFNs, IFN-mediated regulation of LINC01871 expression was examined in the Kasumi-3 myeloid cell line. While LINC01871 expression was not modulated by type I IFN stimulation, it was robustly responsive to IFNγ treatment. Since LINC01871 was expressed in T cells and implicated in T cell pathways, responses to TCR signaling pathways were characterized in HSB2 cells or purified primary human T cells treated with PMA/I. In both cell types, LINC01871 exhibited a prolonged decrease in expression that was abrogated by concurrent treatment with FK506, indicating that LINC01871 is regulated by calcineurin signaling. Treatment of primary human T cells with anti-CD3/CD28 to mimic true TCR engagement resulted in a modest decrease of LINC01871 expression at early time points, followed by an increase in expression with longer stimulation (2d). Conclusion Our findings suggest that LINC01871 is a potential mediator of the dysregulated T cell inflammatory response pathways implicated in SjD pathogenesis. LINC01871 influences the expression of many important immune cell genes and growth factors, is inducible by IFNγ, and is regulated directly by calcineurin signaling and TCR ligand engagement. Although LINC01871 functions are still unknown, observed LINC01871 overexpression in whole blood of SjD cases and after prolonged TCR stimulation in primary human T cells suggests that it could be a biomarker of SjD. References [1]Joachims, et al. Annals of the Rheumatic Diseases 2020;79:90. Disclosure of Interests Michelle L Joachims: None declared, Bhuwan Khatri: None declared, Chuang Li: None declared, Kandice L Tessneer: None declared, John Ice: None declared, Anna M Stolarczyk: None declared, Nicolas Means: None declared, Kiely Grundahl: None declared, Stuart Glenn: None declared, Jennifer Kelly: None declared, David Lewis: None declared, Lida Radfar: None declared, Donald Stone: None declared, Joel Guthridge: None declared, Judith A. James: None declared, R Hal Scofield: None declared, Graham B Wiley: None declared, Jonathan Wren: None declared, Patrick M Gaffney: None declared, Courtney Montgomery: None declared, Kathy Sivils Employee of: Current employee of Janssen., Astrid Rasmussen: None declared, A Darise Farris: None declared, Indra Adrianto: None declared, Christopher Lessard: None declared